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Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer


Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescence-associated heterochromatic foci (SAHF). Here we show that in human fibroblasts resistant to premature p16INK4a induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogene-induced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogene-expressing cells increase DDR signalling and lead to apoptosis. In vivo, a histone deacetylase inhibitor (HDACi) causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours.

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Figure 1: SAHF are preferentially formed on oncogene-induced senescence in human normal fibroblasts.
Figure 2: SAHF formation requires DNA replication and is dependent on ATR.
Figure 3: Increased heterochromatin in DDR-deficient oncogene-expressing cells is compatible with cellular proliferation.
Figure 4: E2F target genes are not repressed by heterochromatin induction in DDR-deficient oncogene-expressing cells.
Figure 5: Increased heterochromatin is retained in human tumours in vivo in different stages of cancer progression.
Figure 6: SAHF and DDR markers coexist in OIS cells but do not colocalize.
Figure 7: Heterochromatin induction restrains oncogene-induced DDR signalling.
Figure 8: Oncogene-induced heterochromatin formation prevents apoptosis by restraining oncogene-induced DDR signalling.


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We thank M. Fumagalli for providing fibroblasts undergoing senescence following telomere shortening and for editing the manuscript; A. Oldani and D. Parazzoli from IFOM Imaging Unit for help with imaging; qRT–PCR and Cell Biology Units for support; S. Vultaggio for help with tumour xenograft generation and M. Romanenghi for technical assistance with ChIP. We thank O. Fernandez-Capetillo for pLKO.1 shATR; G. Smith for KU55933 (KuDOS Pharmaceuticals Ltd.). G. Manfioletti for sharing HMGA antibodies; P.P. Di Fiore for support; U. Herbig, B. Amati and M. Foiani for critical reading of the manuscript and all F.d'A.d.F. lab members for discussion and feedback throughout this work. M.L. and V.G. are funded by the European Commission (FP7-GENICA project). W.C.H. is supported in part by an U.S. NIH/NIA grant (ROI AG023145). The S.M. laboratory is supported in part by the European Union (Epitron). The F.d'A.d.F laboratory is supported by AIRC (Associazione Italiana per la Ricerca sul Cancro), the European Community's 7th Framework Programme (FP7/2007-2013) under grant agreement number 202230 (Genomic instability and genomic alterations in pre-cancerous lesions and/or cancer; GENINCA), HFSP (Human Frontier Science Program), AICR (Association for International Cancer Research), EMBO Young Investigator Program and Telethon.

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M.D. performed immunofluorescence microscopy experiments in Figs 2a and 7a and Supplementary Fig. S2a, and provided technical assistance for cell culture experiments. V.M. performed immunofluorescence microscopy experiments in Figs 2b and 6b and provided technical assistance for cell culture experiments. M.L. and V.G. generated and analysed data and images in Fig. 5a–g and Supplementary Fig. S3. O.A.B. generated data in Fig. 8b, c and Supplementary Fig. S7f, g. G.G. generated data in Fig. 4b and Supplementary Fig. S2l. R.d.Z. and C.M. generated and analysed data in Fig. 8d, e, f and Supplementary Fig. S8a. E.M. contributed to imaging analysis. G.d'A. performed statistical analysis on an imaging dataset. W.C.H. provided shRNA vectors and Ras transformed fibroblasts (ELR) and edited the manuscript. S.M. supervised O.A.B., R.d.Z. and C.M. work, contributed to experimental plan and edited the manuscript. R.D.M. and G.S. generated data of all remaining figures, assembled the manuscript and contributed to experimental design and manuscript writing. F.d'A.d.F. planned and supervised the project and wrote the manuscript.

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Correspondence to Fabrizio d'Adda di Fagagna.

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S.M. has stocks in Genextra Spa, a biopharmaceutical company that is currently developing HDAC inhibitors for cancer therapy.

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Di Micco, R., Sulli, G., Dobreva, M. et al. Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol 13, 292–302 (2011).

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